Technetium-99m labeled peptides for imaging comprising a single thiol moiety

ABSTRACT

The invention elates to radiolabeled imaging of a mammalian body. The invention in particular provides for reagents labeled with technetium-99m for such imaging. The invention provides peptides which bind Tc-99m and which can be targeted to specific sites within a mammalian body.

This is a divisional of application Ser. No. 07/807,062, filed Nov. 27,1991, now U.S. Pat. No. 5,443,815, issued Aug. 16, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to technetium-99m (Tc-99m) labeled reagents, methodsand kits for making such reagents, and methods for using such reagents.In particular, the invention relates to Tc-99m labeled peptidescomprised of between 4 and 100 amino acid residues and covalently linkedto a radioisotope complexing group.

2. Description of the Prior Art

The use of chelating agents for radiolabeling polypeptides is known inthe prior art.

Byrne et al., U.S. Pat. No.4,434,151 describe homocysteine thiolactonebifunctional chelating agents that can couple radionuclides to terminalamino-containing compounds capable of localizing in an organ or tissueto be imaged.

Fritzberg, U.S. Pat. No.4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Gansow et al., U.S. Pat. No.4,472,509 teach methods of manufacturing andpurifying metal chelate-conjugated monoclonal antibodies.

Byrne et al., U.S. Pat. Nos. 4,571,430 and 4,575,556 describe novelhomocysteine thiolactone bifunctional chelating agents for chelatingradionuclides that can couple radionuclides to terminal amino-containingcompounds capable of localizing in an organ or tissue to be imaged.

Nicolotti et al., U.S. Pat. No.4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies. This reference describes compounds such asS-benzoylmercaptoacetylglycylglycylglycine.

European Patent Application No. 84109831.2 describes technetiumchelating complexes of bisamido-bisthio-ligands and salts thereof, usedprimarily as renal function monitoring agents.

European Patent Application No. 86100360.6 describes dithiol, diamino,or diamidocarboxylic acids or amine complexes useful for makingtechnetium imaging agents.

European Patent Application No. 88104755.9 describes various S-protectedmercaptoacetylglycylglycine chelating groups bound to large proteinssuch as antibodies.

Albert et al., UK Patent Application No. 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ Invia a chelating group bound to the amino terminus.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling synthetic peptide fragments via a set of organicchelating molecules.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Davison et al., Inorg. Chem. 20: 1629-1632 (1981) disclose oxotechnetiumchelate complexes.

Fritzberg et al., J. Nucl. Med. 23: 592-598 (1982) disclose a technetiumchelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.

Byrne and Tolman, J. Nucl. Med. 24: P126 (1983) disclose a bifunctionalthiolactone chelating agent for coupling Tc-99m to biological molecules.

Bryson et al., Inorg. Chem. 27: 2154-2161 (1988) describe thiolateligands for complexing with technetium.

Bryson et al., Inorg. Chem. 29: 2948-2951 (1990) describe thiolateligands for complexing with technetium.

Kwekkeboom et al., J. Nucl. Med. 32: 981 (1991) Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., Abstract LM10, 12th American Peptide Symposium (1991)describe uses for ¹¹¹ In-labeled diethylenetriaminopentaaceticacid-derivatized somatostatin analogues.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶Re. The sensitivity of imaging methods using radioactively-labeledpeptides is much higher than other techniques known in the art, sincethe specific binding of the radioactive peptide concentrates theradioactive signal over the cells of interest, for example, tumor cells.

Methods for labeling peptides and polypeptides with Tc-99m have beendisclosed in the prior art.

Dean, co-pending U.S. patent application Ser. No. 07/653,012 teachesreagents and methods for preparing peptides comprising a Tc-99mchelating group covalently linked to a specific binding peptide forradioimaging in vivo, and is hereby incorporated by reference.

Fritzberg, U.S. Pat. No.4,444,690 describes a series oftechnetium-chelating agents based on2,3-bis(mercaptoacetamido)propanoate.

Gansow et al., U.S. Pat. No.4,472,509 teach methods of manufacturing andpurifying Tc-99m chelate-conjugated monoclonal antibodies.

Reno and Bottino, European Patent Application No. 87300426.1 discloseradiolabeling antibodies with Tc-99m.

Bremer et al., EPC Application No. 87118142.6 disclose organ-specificradioimaging using Tc-99M radiolabeled proteins.

Pak et al., European Patent Application No. WO 88/07382 disclose amethod for labeling antibodies with Tc-99m.

Goedemans et al., PCT Application No. WO 89/07456 describe radiolabelingproteins using cyclic thiol compounds, particularly on 2-iminothiolaneand derivatives.

Schochat et al., PCT Application No. WO 89/09405 disclose directradiolabeling of proteins comprised of at least one "pendent" sulfhydrylgroup.

Dean et al., PCT Application No. WO 89/12625 describe bifunctionalcoupling agents for Tc-99m labeling of proteins or peptides.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Stuttle, PCT Application No. WO 90/15818 describes Tc-99m labeling ofRGD-containing oligopeptides.

Rhodes, Sem. Nucl. Med. 4: 281-293 (1974) teaches the labeling of humanserum albumin with Tc-99m.

Khaw et al., J. Nucl. Med. 23: 1011-1019 (1982) disclose methods forlabeling biologically active macromolecules with Tc-99m.

Byrne and Tolman, supra, disclose a bifunctional thiolactone chelatingagent for coupling Tc-99m to biological molecules.

Knight et al., Abstract #209, 37th Annual Meeting, Society for NuclearMedicine (1990) describe thrombus imaging with Tc-99m labeled peptides.

Cox et al., Abstract, 7th International Symposium on Radiopharmacology,p. 16, 1991, disclose the use of ¹³¹ I- and ¹¹¹ In-labeled somatostatinanalogues in radiolocalization of endocrine tumors in vivo byscintigraphy.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic imaging agents that areradioactively-labeled peptides. The peptides of the invention arecomprised of between 4 and 100 amino acid residues, covalently linked toa radioisotope complexing group wherein the complexing group binds aradioisotope.

Preferred embodiments of the present invention are peptides that arecovalently linked to radioisotope complexing groups comprising a thiolmoiety having the following structure:

    A--CH(B)-- C(RR')!.sub.n --X

wherein A is H or HOOC; B is H, SH or NHR", where R" is H or loweralkyl; X is SH or NHR", where R" is H or lower alkyl; R and R' areindependently H or lower alkyl; n is 0, 1 or 2; and: 1. where B is NHR",where R" is H or lower alkyl, X is SH and n is 1 or 2; and 2. where X isNHR", where R" is H or lower alkyl, B is SH and n is 1 or 2; 4. where Bis H, A is HOOC, X is SH and n is 0 or 1; and wherein the thiol moietyis in the reduced form.

The invention encompasses peptides for labeling with Tc-99m and imagingtarget sites within a mammalian body comprising between 4 and 100 aminoacid residues, covalently linked to a radioisotope complexing groupwherein the complexing group binds a radioisotope. The inventionencompasses methods for making such peptides covalently linked to aradioisotope complexing group. The invention also includes Tc-99mcomplexes and methods for preparation of such Tc-99m complexes andmethods for using the Tc-99m complexes to image target sites within amammalian body.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides Tc-99m labeled peptides for imagingtarget sites within a mammalian body that comprise between 4 and 100amino acid residues and are covalently linked to a radioisotopecomplexing group wherein the complexing group binds a radioisotope.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Other radionuclides known inthe prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (forexample, ¹²⁵ I).

Peptides of the present invention can be chemically synthesized invitro. Peptides of the present invention can generally advantageously beprepared on an amino acid synthesizer. The peptides of this inventioncan be synthesized wherein the complexing group is covalently linked tothe peptide during chemical in vitro synthesis, using techniques wellknown to those with skill in the art. Such peptides covalently-linked tothe complexing group upon synthesis are advantageous because specificsites of covalent linkage can be determined therein.

In forming a complex of radioactive technetium with the peptides of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the peptides of this invention in thepresence of a reducing agent; in a preferred embodiment, the reducingagent is stannous chloride. In an additional preferred embodiment, thereducing agent is a solid-phase reducing agent. Complexes and means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of thepeptides of the invention that are to be labeled and a sufficient amountof reducing agent to label the peptide with Tc-99m. Alternatively, thecomplex may be formed by reacting the peptides of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts. The reaction ofthe peptides of this invention with Tc-pertechnetate or preformed Tc-99mlabile complex can be carried out in an aqueous medium at roomtemperature. The anionic complex which has a charge of -1! is formed inthe aqueous medium in the form of a salt with a suitable cation such assodium cation, ammonium cation, mono, di- or tri-lower alkyl aminecation, etc. Any conventional salt of the anionic complex with apharmaceutically acceptable cation can be used in accordance with thisinvention.

In another embodiment of the present invention, the peptides of theinvention that are to be labeled are reduced prior to labeling byincubating the peptides with a reducing agent. In a preferredembodiment, the reducing agent is stannous chloride. In an additionalpreferred embodiment, the reducing agent is a solid-phase reducingagent. The pre-reduced peptide is then labeled by reaction with a Tc-99munder reducing conditions or with pre-reduced Tc-99m or Tc-99m complex.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. The peptides of the inventioncan be chemically synthesized using methods and means well-known tothose with skill in the art and described hereinbelow. Peptides thusprepared are comprised of between 4 and 100 amino acid residues, and arecovalently linked to a radioisotope complexing group wherein thecomplexing group binds a radioisotope. An appropriate amount of thepeptide is introduced into a vial containing a reducing agent, such asstannous chloride or a solid-phase reducing agent, in an amountsufficient to label the peptide with Tc-99m. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. Technetium-labeled peptidesaccording to the present invention can be prepared by the addition of anappropriate amount of Tc-99m or Tc-99m complex into the vials andreaction under conditions described in Example 2 hereinbelow.

Radioactively labeled peptides provided by the present invention areprovided having a suitable amount of radioactivity. In forming theTc-99m radioactive anionic complexes, it is generally preferred to formradioactive complexes in solutions containing radioactivity atconcentrations of from about 0.01 millicurie (mCi) to 100 mCi per ml.

Technetium-labeled peptides provided by the present invention can beused for visualizing organs such as the kidney for diagnosing disordersin these organs, and tumors, such as gastrointestinal tumors, myelomas,small cell lung carcinoma and other APUDomas, endocrine tumors such asmedullary thyroid carcinomas and pituitary tumors, brain tumors such asmeningiomas and astrocytomas, and tumors of the prostate, breast, colon,and ovaries can also be imaged. In accordance with this invention, thetechnetium-labeled peptides or anionic complexes either as a complex oras a salt with a pharmaceutically acceptable cation are administered ina single unit injectable dose. Any of the common carriers known to thosewith skill in the art, such as sterile saline solution or plasma, can beutilized after radiolabeling for preparing the injectable solution todiagnostically image various organs, tumors and the like in accordancewith this invention. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 ml toabout 10 ml. After intravenous administration, imaging of the organ ortumor in vivo can take place in a matter of a few minutes. However,imaging can take place, if desired, in hours or even longer, afterinjecting into patients. In most instances, a sufficient amount of theadministered dose will accumulate in the area to be imaged within about0.1 of an hour to permit the taking of scintiphotos. Any conventionalmethod of scintigraphic imaging for diagnostic purposes can be utilizedin accordance with this invention.

The technetium-labeled peptides and complexes provided by the inventionmay be administered intravenously in any conventional medium forintravenous injection such as an aqueous saline medium, or in bloodplasma medium. Such medium may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. Among the preferred media are normal saline and plasma.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature. Crude peptides were purified by preparative highpressure liquid chromatography (HPLC) using a Waters Delta Pak C18column and gradient elution using 0.1% trifluoroacetic acid (TFA) inwater modified with acetonitrile. Acetonitrile was evaporated from theeluted fractions which were then lyophilized. The identity of eachproduct was confirmed by fast atom bombardment mass spectroscopy(FABMS).

EXAMPLE 2 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 1 was dissolved in 0.1 ml of0.05M potassium phosphate buffer (pH 7.4). Tc-99m gluceptate wasprepared by reconstituting a Glucoscan vial (E. I. DuPont de Nemours,Inc.) with 1.0 ml of Tc-99m sodium pertechnetate containing up to 200mCi and allowed to stand for 15 minutes at room temperature. 25 ul ofTc-99m gluceptate was then added to the peptide and the reaction allowedto proceed at room temperature for 30 min and then filtered through a0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using a Vydak218TP54 analytical column (RP-18, 5 micron, 220×4.6 mm) and eluted withthe following gradient: 100% A (0.1% TFA in H₂ O) to 100% B (CH₃ CN:H₂O:TFA, 70:30:0.1) over 10 minutes at a flow rate of 1.2 ml/min; and thenheld at the 100% B solution for 5 minutes. Radioactive components weredetected by an in-line radiometric detector linked to an integratingrecorder. Tc-99m gluceptate and Tc-99m sodium pertechnetate elutebetween 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 1 using the method described herein.

                                      TABLE I                                     __________________________________________________________________________                        FABMS MH+                                                                             Radiochemical                                                                        HPLC Rt                                    Peptides Prepared and Radiolabeled                                                                Found/Theoretical                                                                     Yield  (min)                                      __________________________________________________________________________    YRALVDTLKFVTQAEGAKC.NH.sub.2 (SEQ. ID                                                             2113/2113.1                                                                           92%      11-15.5                                  NO: 1)                                                                        GRGDGGC (SEQ. ID NO: 2)                                                                           769/768.3                                                                             98%    13-15                                      maGGGRGDF.sup.a (SEQ ID NO: 3)                                                                    739/739.3                                                                             98%    13-15                                      PenGGGRALVDTLK.NH.sub.2 .sup.b (SEQ. ID NO: 4)                                                    1216.3/1216.7                                                                         98%    14.8                                       maGGGGRALVDTLK.NH.sub.2 (SEQ. ID NO: 4)                                                           1160/1160.5                                                                           97%    14.3                                       mmpGGGRALVDTLK.NH.sub.2 .sup.c (SEQ. ID NO: 4)                                                    1187.4/1186.7                                                                         98%    14.9-15.5                                  __________________________________________________________________________     .sup.a ma = mercaptoacetic acid                                               .sup.b Pen = Lpenicillamine                                                   .sup.c mmp = 2mercapto-2-methylpropionic acid                            

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrArgAlaLeuValAspThrLeuLysPheValThrGlnAlaGluGly                              151015                                                                        AlaLysCys                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyArgGlyAspGlyGlyCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyGlyGlyArgGlyAspPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlyGlyGlyArgAlaLeuValAspThrLeuLys                                             1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A compound comprising:a) a peptide having between4 and 100 amino acid residues; and b) covalently linked to the peptide,a radioisotope complexing group comprising a single reduced thiol moietythat is:

    A--CH(B)-- C(RR')!.sub.n --X

wherein A is H or HOOC; B is H, SH, or NHR", where R" is H or loweralkyl; X is SH or NHR", where R" is H or lower alkyl; R and R' areindependently H or lower alkyl; n is 0, 1 or 2; and where B is NHR", Xis SH and n is 1 or 2; where X is NHR", B is SH and n is 1 or 2; where Bis H, A is HOOC, X is SH and n is 0 or 1; wherein the compound iscapable of imaging target sites within a mammalian body when labeledwith technetium-99m.
 2. The compound according to claim 1 wherein thepeptide and the complexing group are covalently linked through one ormore amino acids.
 3. The compound according to claim 1 wherein thepeptide is chemically synthesized in vitro.
 4. The compound according toclaim 3 wherein the peptide is synthesized by solid phase peptidesynthesis.
 5. The compound according to claim 3 wherein the complexinggroup is covalently linked to the peptide during in vitro chemicalsynthesis.
 6. The compound according to claim 4 wherein the completinggroup is covalently linked to the peptide during solid phase peptidesynthesis.